1. Field of the Invention
The present invention relates generally to the fields of cell biology and molecular biology. More particularly, it concerns methods and compositions to prepare a biological sample, such as a fresh or frozen tissue sample, to enhance the preservation of biological macromolecules in the sample. Methods and compositions of the invention include one or more water-miscible solvents that have a freezing temperature below the freezing temperature of water. Such compositions make frozen tissue samples easier to process for the removal of biological macromolecules while protecting them from degradation.
2. Description of Related Art
The field of molecular biology has a high demand for RNA samples extracted from isolated animal and plant tissues, as well as cultured cells. These samples are analyzed to ascertain changes in the steady-state expression levels of mRNAs in response to disease or experimental stress between different samples. The analysis is performed by a variety of methods, including reverse-transcription polymerase chain reaction (RT-PCR®), Northern blot hybridization, nuclease protection assays, and, more recently, gene array analysis.
For studies of the purified RNA samples to be meaningful, they must accurately reflect the RNA population in the cells at the time of isolation. This means that immediately after the sample is obtained, measures should be taken so that no RNA is created or destroyed in the interval from sample collection to RNA purification. From both a theoretical and practical viewpoint, the degradation of RNA is the more problematic process, as synthesis is a delicately balanced process that tends to become disrupted with the shift in metabolism that occurs with tissue excision. The removal of energy sources that occurs with sample collection shifts the balance to primarily catabolic processes, which results in the inappropriate degradation of RNA by components normally meant to remove older or inappropriate RNA molecules. The process of tissue collection (presumably by excising it from its source) represents the first of several stages where degradation of RNA may occur.
After a sample is collected, it is conveyed to an appropriate homogenization medium, which contains denaturants (e.g., guanidinium salts, reductants, and detergents) that inactivate RNA degradation enzymes and disrupt complexes on contact to release RNA into solution. Once in this medium, the tissue usually has to be mechanically disrupted to break up insoluble tissue components and allow full penetration of the denaturants in which it is homogenized. The final isolation of RNA from this homogenate usually entails an actual chemical extraction of RNA from its cellular milieu such as: absorption of the RNA to a solid support, precipitation of the RNA from the denaturation solution, or extraction of the protein component of the mixture using organic solvents such as phenol and chloroform. A great deal of work has been done to assemble homogenization solutions that work quickly upon tissue disruption and do not allow any biochemical processes to occur during the extraction that might alter mRNA and protein levels. However, these do not address the other stages at which degradation may occur; specifically the time required to convey the tissue sample to homogenization solution and the time in this solution prior to physical disruption of the tissue.
In some cases the sample isolation must be performed away from a laboratory setting, and the delay between sample collection and RNA isolation is quite extended. Alternatively, sometimes it is not desirable to immediately process the tissue and extract RNA. In these instances, the preferred method for stopping cellular metabolism is by quick-freezing the tissue samples, usually by immersion in liquid nitrogen. The downstream processing required to extract the RNA from samples frozen in this manner can be quite laborious.
It is well known that for intact RNA to be obtained from frozen tissue samples, it must be extracted with a minimum of thawing taking place (see Rapley and Manning, 1998). It is thought that ice damages the normal barriers formed between cellular compartments, so that nucelolytic enzymes in degradosomes, cytoplasmic vesicles, and extracellular regions are allowed ectopic access to cytoplasmic and nuclear RNA. To minimize this effect, the preferred method to process frozen tissue is to grind it to a fine powder while maintaining its frozen state, using a specialized refrigerated pulverizing machine or a mortar and pestle kept constantly frozen with dry ice or liquid nitrogen. Since few researchers have access to refrigerated pulverizers, the latter process is routinely used. This is time- and labor-intensive, requiring pre-chilling the mortar and pestle slowly in a −80° C. freezer (plunging in liquid nitrogen tends to crack the ceramic), donning insulated gloves to handle the chilled pestle, and continuously adding liquid nitrogen to the mortar during the process of grinding. The grinding process is laborious, often taking 5 minutes for a one gram sample. If several samples need to be processed in this fashion, a separate mortar and pestle must be used for each, or the apparatus must be thawed, thoroughly cleaned, and re-chilled prior to the next use.
Once pulverized, the powder can then be poured from the frozen mortar into a chilled container for further storage or directly into cold homogenization solution and immediately homogenized to ensure rapid penetration of protein denaturants and inactivation of endogenous nuclease activity. Once in the denaturation solution, the sample can be further homogenized by mechanical blenders, rotor-stator homogenizers, or shear-type homogenizers which pass the solution through a thin space between a plunger and reinforced test tube several times, often while mechanically rotating the plunger. Transfer to the homogenization vessel can be problematic. Since the ambient air is substantially warmer than the sample (even if the procedure is performed in a cold room), atmospheric moisture tends to condense on the sample, causing the powder to clump and stick to the side of receiving vessels, creating masses of tissue thawing out before being homogenized or even submerged in the homogenization solution.
As an alternative, some researchers will grind tissue with frozen homogenization solution, then thaw the powder after the tissue and homogenization solution are thoroughly ground and blended together. With any of these homogenization procedures, a finite amount of time is required to process the sample to uniformity, and during this time, there is the potential for degradation. Thus, with current techniques, there is the dual problem of thawing on transfer or in homogenization solution and the laborious process of grinding tissue at −80° C. or lower (liquid nitrogen boils at −195.8° C.).
The prevalent use of cryostorage for archiving tissues makes any method for the alleviation of the chore of pulverizing into powder before the actual extraction of RNA extremely desirable.
There is currently no procedure or product specified to maintain the intactness of the RNA in a frozen sample while transitioning the sample to a non-brittle state. The term ‘frozen’ refers to a state where the water contained in the tissue sample is physically present in a solid state. Obviously frozen tissue is a solid mass, and therefore extremely recalcitrant to having solutions diffuse into it. Although tissues can be saturated with preservative solutions at warmer temperatures (e.g., RNAlater™, Ambion, Inc., Austin Tex.), aqueous solutions will be solid at the temperature used to store frozen samples (usually −80° C. or colder). There are reports that solutions of ethanol (U.S. Pat. No. 5,256,571; Safneck et al., 2001, Esser et al., 1995) and acetone (Fukatsu, 1999) provide limited protection for RNA when used to saturate tissues above 0° C. (usually 4° C. or room temperature), although, these are not widely used.
Thus, there is a need for improved methods and compositions that render frozen biological samples tractable for immediate homogenization, as well as for methods and compositions that preserve as many macromolecules from the sample as possible. A method enabling the use of tissue pieces instead of powdered tissue would greatly simplify handling and processing.